标题：新生鼠原代肺泡Ⅱ型上皮细胞的分离、纯化与鉴定

      作者：张梦颖，郭瑞敏，孙燕妮    上海中医药大学附属普陀医院中心实验室，上海 200062

      卷次： 2020年31卷2期

      【摘要】 目的 从新生SD大鼠体内分离提取肺泡Ⅱ型上皮细胞，为肺部相关疾病提供体外模型 方法 胰蛋白酶、胶原酶Ⅰ消化分离细胞，差速贴壁法和大鼠 IgG免疫黏附法纯化。采用倒置显微镜、透射电镜、共聚焦显微镜以及BCIP/NBT碱性磷酸试剂盒对细胞进行观察和鉴定。结果 采取胰蛋白酶、胶原酶消化得到细胞，活力为(94.82±1.64)%，纯度为(95.96±0.90)%。倒置显微镜观察细胞内呈立方形，岛状生长，胞浆内有黑色颗粒；透射电镜下观察到特异性板层小体；共聚焦显微镜下可看到表面活性蛋白C表达；BCIP/NBT碱性磷酸试剂盒鉴定后可观察到细胞胞浆内有深蓝色颗粒。结论 用胰蛋白酶、胶原酶 I消化可得到高纯度的肺泡Ⅱ型上皮细胞，可满足体外实验的需要。
      【关键词】 肺泡二型上皮细胞；原代分离；纯化；原代培养；新生鼠
      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2020）02—0137—04

Isolation, purification, and identification of primary alveolar type Ⅱ epithelial cells from newborn rats.
ZHANGMeng-ying, GUO Rui-min, SUN Yan-ni. Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200062, CHINA
【Abstract】 Objective Alveolar type Ⅱ epithelial cells were isolated from newborn SD rats to provide an invitro model of pulmonary diseases. Methods The cells were isolated by digestion of trypsin and collagenase Ⅰ, andpurified by differential adherent method and IgG immunoadhesion method in rats. Cells were observed and identifiedby inverted microscope, transmission electron microscope, confocal microscope and BCIP/NBT basic phosphate kit.Results The cells were digested with trypsin and collagenase, and the activity and purity were (94.82 ± 1.64)% and(95.96±0.90)%, respectively. Under inverted microscope, the cells were cubic and island-like, with black granules in thecytoplasm. Lamellar bodies were revealed by ultrastructual examination under electron microscope. The expression ofsurfactant protein C in alveolar typeⅡ epithelial cells was observed under confocal microscope. After BCIP/NBT basicphosphate kit was identified, dark blue granules were observed in the cytoplasm of the cells. Conclusion High-purityalveolar typeⅡ epithelial cells can be obtained by digestion with trypsin and collagenaseⅠ, which can meet the needsof in vitro experiments.
      【Key words】 Alveolar epithelial typeⅡ; Primary isolation; Purification; Primary culture; Neonatal rat